Receptor tyrosine kinases (RTKs) mediate intercellular signals which are essential for the development and maintenance of cells of multicellular organisms. The minimal domain structure of RTKs consists of an extracellular ligand-binding domain structure, a single transmembrane helix, and a cytoplasmic tyrosine kinase (TK) domain.
c-Met (mesenchymal-epithelial transition factor) is a cell surface, disulphide linked heterodimeric RTK encoded by the c-met protooncogene. The c-Met receptor is also referred to as hepatocyte growth factor receptor (HGFR). It is the receptor for hepatocyte growth factor/scatter factor (HGF/SF) which is a large polypeptide growth factor discovered as a protein causing dispersion of epithelial colonies and cell migration (SF) and as a liver mitogen (HGF).
The c-Met receptor is synthesized as a 170 kDa precursor which is glycosylated and proteolytically cleaved in the post-Golgi compartment into an extracellular 50 kDa α-chain and an extracellular/intracellular 145 kDa β-chain that contains the TK domain. The chains remain associated after cleavage due to a disulfide linkage resulting in a heterodimeric molecule. The HGF/SF and heparin-binding sites of c-Met are contained within a large N-terminal domain spanning the alpha chain (amino acids 25-307) and the first 212 amino acids of the beta chain (amino acids 308-519). Within residues 520-561 is a cysteine-rich domain. Neither this domain, nor the C-terminal half of c-Met (amino acids 562-932) bind HGF/SF or heparin directly.
The extracellular portion of c-Met contains a region of homology to semaphorins (Sema domain, which includes the full alpha chain and the N-terminal part of the beta chain of c-Met), a cysteine-rich c-Met related sequence (MRS) followed by glycine-proline-rich (G-P) repeats, and four immunoglobulin-like structures. The intracellular region of c-Met contains three region: (i) a juxtamembrane segment that contains (a) a serine residue (Ser 985) that, when phosphorylated by protein kinase C or by Ca2+ calmodulin-dependent kinases downregulates the receptor kinase activity; and (b) a tyrosine (Tyr 1003) that binds the ubiquitin ligase Cbl responsible for Met polyubiquitination, endocytosis and degradation; (ii) a tyrosine kinase domain that, upon receptor activation, undergoes transphosphorylation on Tyr1234 and Tyr1235; and (iii) the C-terminal region, which comprises two crucial tyrosines (Tyr1349 and Tyr1356) inserted in a degenerate motif that represents a multi-substrate docking site capable of recruiting several down stream adaptors containing Src homology-(SH2) domains. A detailed review of the structure can be found in, Gentile, A et al., (2008).
c-Met Expression and Signaling
Typically c-Met expression is found in epithelial derived cells while HGF/SF is expressed in the surrounding mesenchyme. During development, c-Met is required for proper development of the placenta, liver, kidney, neuronal and skeletal muscle. In adults, c-Met expression plays a role in hematopoiesis, including B-cell development during antigen selection, and is upregulated during tissue injury.
Upon HGF binding, c-Met homodimerises which leads to the activation of its TK domain, as well as autophosphorylation of several tyrosine residues including the C-terminal residues Y1349 and Y1356. Phosphorylated Y1349 and Y1356 form a multi-substrate docking site (Y1349VHVXXXY1356VNV) (SEQ ID NO:43) capable of binding several adaptor proteins to initiate downstream signaling, commonly using the PI3K/Akt and Ras/MAPK pathways (Eder J P et al., 2009, and Birchmeier C et al., 2003). The multi-substrate docking site is an absolute requirement for c-Met signaling.
The juxtamembrane domain of c-Met also plays a regulatory role. Phosphorylation of Y1003 in this domain is involved in c-Met downregulation as it binds proteins such as the E3 ubiquitin ligase, CBL. Binding of CBL also leads to the recruitment of the endophilin-CIN85 complex, resulting in c-Met internalization and degradation (Ma et al., 2003). Phosphorylation of S975 in the juxtamembrane domain is also involved in c-Met internalization and has been shown to be phosphorylated by protein kinase C and Ca2+-calmodulin-dependent kinase. The internalization of c-Met is necessary for ERK signaling.
In addition to ligand activation of c-Met, there is also signal induction from cross-talk between c-Met and other receptors. In tumor cells, c-Met co-immunoprecipitates with EGFR regardless of the presence of their ligands.
Role of c-Met in Cancer
The c-Met receptor tyrosine kinase is involved in multiple pathways linked to cancer, such as cell migration, invasion, proliferation and angiogenesis, and is upregulated in a large number of cancers (Christensen, J G et al., (2005) Cancer Let 225: 1-26; Jiang, W G et al., (2005) Crit Rev Oncol Hematol 53:35-69). c-Met was first identified as an oncogene in 1984 and is among the most frequently expressed oncogenes in human cancer. C-Met alterations or deregulation (mutations, altered expression, amplification) has been associated with many types of human cancers, including kidney, liver, stomach, colon, breast, brain, prostate, ovarian, lung, bladder, head and neck, thyroid (Birchmeier, C et al., (2003) Cell Biol 4:915-925; Corso S et al., (2005) Trends Mol Med 11:284-92; Christensen J G., et al (2005) Cancer Lett 225:1-26; Salgia, R (2009) Semin Oncol 36(2 Suppl 1) S52-58; Engelman J A., et al (2007) Science 316:1039-1043; vai.org/vari/met and cancer). Genetic alterations which generate ligand-independent c-Met mutants have been found in both hereditary and sporadic papillary renal cell carcinomas and involve mutations in the tyrosine kinase domain of c-Met (Schmidt L et al., (1997) Nat Genet 16:68-73; Schmidt L et al., (1999) Oncogene 18:2343-50; Dharmawardana P G et al., (2004) Curr Mol Med 4:855-68). Missense mutations in c-Met (primarily in the kinase domain) have also been identified in ovarian cancer, childhood hepatocellular carcinoma, metastatis head and neck squamous cell carcinomas, and gastric cancer (Gentile et al., 2008; Ma et al., 2003). In melanoma and thoracic malignancies such as small cell lung cancer and mesothelioma, c-Met mutations clustered predominantly in the SEMA and juxtamembrane domains (Ma P C et al., 2003, Puri N et al., 2007 and Jagadeeswaran R et al., 2006).
The most common genetic alteration involving c-Met is gene amplification leading to c-Met over-expression. c-Met overexpression has been found in a large number of human tumors including breast, gastric, cervix, hepatocellular, brain and head and neck cancers (www.vai.org/met) and may also lead to ligand-independent kinase activation. Approximately 25% of ovarian cancers and 11% of gliomas express high levels of c-Met. Most often in cancer, c-Met activation occurs via a ligand-dependent mechanism. This stimulation is often autocrine as is typically seen in glioblastoma and multiple myeloma.
The HGF:c-Met signaling axis has an important role in the initiation and progression of several aggressive cancers such as glioblastoma multiforme (GBM), a lethal tumor of the brain which is refractory to currently available therapies. While the c-met gene is amplified in approximately 4% of GBM (McLendon R et al., 2008) it is over-expressed in high grade GBM and often co-expressed with HGF. Over-expression of c-Met in GBM reduces patient progression-free and overall survival times (Doo-Sik Kong et al., 2009).
Targeting c-Met
c-Met is one of the most frequently genetically altered or otherwise dysregulated receptor tyrosine kinases in advanced human cancer and accordingly has been intensely investigated as a therapeutic target with several classes of agents being developed as novel therapeutics. These include small molecular weight tyrosine kinase inhibitors (TKI's), which prevent the activation of c-Met by acting as ATP-binding competitors for the TK domain. These TKI's have been shown to have anti-tumor activity in both in vitro and in vivo models (reviewed in Stellrecht et al., 2009, Eder J P et al., 2009, Comoglio P M et al., 2008; Tseng J R et al., (2008)), with several candidates currently being evaluated in clinical trials. Other approaches that have been used for targeting c-Met include the use of small interfering RNA (siRNA) and ribozymes which target c-met expression. RTK inhibitors include c-Met specific and more general tyrosine kinase inhibitors, including but not limited to K252a, SU11274 (Sugen). PH-665752 (Pfizer), ARQ197 (ArQule), XL880 (Exelesis), MP470 (SuperGen).
Another class of therapeutics are monoclonal antibodies (mAbs) directed to c-Met or HGF. The present inventors and others have shown that treatment of U87MG GBM xenografts with AMG 102, a fully human neutralizing antibody directed to HGF, significantly inhibits tumor growth (Burgess T et al., 2006, Pillay V et al., 2009). The agonist activity of anti-c-Met antibodies is often due to the antibody inducing receptor dimerisation and thus kinase activation.
DN30, described in Prat M et al., 1991, and Petrelli A et al., 2006 is a monoclonal antibody that binds to the beta chain of c-Met. The antibody behaves as a partial agonist since it induces phosphorylation of the receptor but does not activate the complete set of downstream biological effects of c-Met. The antibody has been found to cause c-Met receptor downregulation through a mechanism involving a double proteolytic cleavage. The c-Met downregulation effect appears to be dependent upon HGF (Petrelli A et al., 2006). The fact that this antibody causes initial activation of the c-Met receptor makes it problematic for therapy.
A further monoclonal MET4 has been described in Knudsen et al., 2009 and WO09/029591. This antibody recognizes an epitope on the alpha chain of human c-Met protein at amino acids 236-242. The antibody was developed as a diagnostic to recognize c-Met by immunohistochemistry in formalin fixed and paraffin embedded tissues. Neither of these disclosures mention anything as to whether this antibody exhibits any anti-tumor activity.
OA-5D5 is a one armed monoclonal anti-Met antibody comprising murine variable heavy and light chain domains and human IgG1 constant domains. This antibody has been described in Ohashi et al., 2000 and WO 06/015371 to Genentech. The OA-5D5 is derived from a fully agonistic anti-c-Met mAb and strongly inhibits the growth of HGF-dependent GBM xenografts in vivo but has no effect on HGF-independent GBM xenografts (Martens T et al., 2006).
More recently the creation of anti-c-Met Fab molecules R13 and R28 has been reported (van der horst E H et al., 2009). These Fab's act together through the initial binding of R13 locking c-Met into a conformation that stabilizes the R28 epitope. R28 then binds c-Met to block HGF binding.
Also, Pfizer CE-355621 antibody has shown efficacy in a U87 MG mouse xenograft model (Tseng et al (2008)).
As evidenced in the literature, there is clearly a strong interest in c-Met as a target for anti-tumor therapy and many different approaches are currently being explored. There is clearly a need for additional specific antibodies that successfully target c-Met on tumor cells in the absence of any receptor agonist activity, or that specifically inhibit c-Met activity, particularly TK activity.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.